Methods and materials for HIV detection

ABSTRACT

Disclosed are immunologically active polypeptides, preferably antibodies or antibody fragments, and most preferably monoclonal antibodies, which are reactive with idiotypes of antibodies to human lymphocyte T4 protein and are reactive with the HIV virion in a manner allowing for in vitro and in vivo neutralization of HIV infectivity and detection of HIV particles in biological fluids. Presently preferred embodiments comprise monoclonal anti-monoclonal-anti-human lymphocyte T4 antibodies produced by new murine hybridoma cell lines JT4C8, JT4C12, JT4C16, JT1-1F3, JT1-1F3-E5, JT1-1D7 and JT2-N15. Also disclosed are active and passive vaccination procedures.

This application is a continuation of application Ser. No. 07/340,710,filed Apr. 20, 1989, now abandoned, which is a division of applicationSer. No. 146,371, filed Feb. 3, 1988, now abandoned, which is acontinuation-in-part of co-pending U.S. patent application Ser. No.064,066, filed Jun. 29, 1987, which is in turn a continuation-in-part ofU.S. patent application Ser. No. 016,282, filed Feb. 19, 1987.

BACKGROUND

The present invention relates generally to methods and materials usefulin the diagnosis and treatment of infection with Human ImmunodeficiencyVirus (HIV). More particularly, the invention relates to immunologicallyactive polypeptides, preferably antibodies or antibody fragments, andmost preferably monoclonal antibodies, which are reactive with idiotypesof antibodies to human lymphocyte T4 protein and also reactive with theHIV virion in a manner allowing for in vitro and in vivo neutralizationof HIV infectivity. Moreover, the invention relates to immunologicallyactive polypeptides useful in vaccine compositions for developingprotective responses to HIV infection.

The state of the art with respect to the epidemiology and immunology ofthe causative agent of AIDS in humans is well summarized in: Laurence,"The Immune System in AIDS", Scientific American, December, 1985, pp.84-93; Gallo, "The First Human Retrovirus", Scientific American,December, 1986, pp. 88-98; Gallo, "The AIDS Virus", Scientific American,January, 1987, pp. 47-56; Levy et al., Science, 225, 840-842 (1984);"Mobilizing Against AIDS", Institute of Medicine, National Academy ofSciences, Harvard University Press (Cambridge, Mass 1986); and, Lane etal., Ann. Rev. Immunol., 3, pp. 477-500 (1985). The role of T4 surfaceglycoprotein (sometimes referred to as "CD4" protein or determinant) ofhuman T lymphocytes in infection by HIV has been extensively studied asrepresented by Dalgleish et al., Nature, 312, pp. 763-767 (1984);Klatzman et al., Science, 312, 767-768 (1984); Klatzman et al., Science,225, pp. 59-62 (1984); McDougal et al., J.Immunol., 135, pp. 3151-3162(1985); and, Maddon et al., Cell, 47, pp. 333-348 (1986). See also,Marrack et al., "The T Cell and Its Receptor", Scientific American,February 1986, pp. 36-45; and, McDougal et al., Science, 231, 382-385(1986).

It has recently been projected that soluble forms of CD4 may havetherapeutic utility in treatment of HIV infection. See, Fisher et al.,Nature, 331, 76-77 (1988); Hussey et al., Ibid, at pp. 78-81; Deen etal., Ibid, at pp. 82-83; and Traunecker et al., Ibid, at pp. 84-86 allof which relate to in vitro neutralization of HIV infectivity by solubleCD4. Among the potential drawbacks to the projected use of soluble CD4therapeutic agents is the known reactivity of CD4 with class II majorhistocompatibility complex ("MHC") molecules present on the surface ofother immune cells including B cells, macrophages and monocytes, leadingto the suggestion that CD4 may need to be modified (e.g., by truncation)prior to attempted therapeutic use.

Numerous reports appear in the literature relating to the potential ofantibodies to neutralize infectivity of HIV in vitro and in vivo andspecifically to attempts at active immunization for the purpose ofdeveloping protective immunity See, e.g., Matthews et al., Proc. Nat'l.Acad. Sci. (USA), 83, pp. 9709-9713 (1986); Norman, "AIDS Therapy A NewPush For Clinical Trials", Science, 230, pp. 1355-1358 (1985) and priorarticles in this series; Newmark, Nature, 324, pp. 304-305 (1986); andnotes appearing in Scientific American, February, 1987, at pages 86-88under the heading, "AIDS: Hope . . . And Warnings", and in NewScientist, December 18, 1986, page 7, under the heading "Can Protein TThwart The AIDS Virus . . . ?". See also, Mitsuya et al., Nature, 325,773-778 (1987); Kennedy et al., Science, 231, 1556-1559 (1986); Chanh etal., EMBO Journal, 5(11), 3065-3071 (1986); Chanh et al., Eur. J.Immunol., 16, 1465-1468 (1986); Putney et al., Science, 4, 1392-1395(1986); and, Matshushita et al., Abstract W.3.2, p.106, "IIIInternational Conference on Acquired Immunodeficiency Syndrome (AIDS),June 1-5, 1987.

Of interest to the background of the present invention are the publishedresults of investigation into the immunological role of anti-idiotypes.See, e.g., Kennedy et al., "Anti-Idiotypes and Immunity", ScientificAmerican, July, 1986, pp. 48-56; Jerne, "The Immune System", ScientificAmerican, July, 1973, pp. 52-60; Marx, "Making Antibodies Without TheAntigens", Science, 228, pp. 162-165 (1985); Finberg et al., CRCCritical Reviews in Immunoloqy, 7, 269-284 (1987); and Kennedy et al.,Science, 232, pp. 220-223 (1986). See, also, Norman, supra, relating toa potential correlation between anti-HIV-immunotherapy and production ofanti-idiotypic antibodies to the HIV surface proteins.

Of particular interest to the background of the present invention is thework reported by McDougal et al., J. Immunoloqy, 137, 2937-2944 (1986)wherein it was noted that: " . . . rabbit anti-idiotypic sera raisedagainst each of four candidate CD4 monoclonal antibodies [OKT4A, OKT4D,OKT4F and Leu3a (sometimes referred to as "anti-Leu3a")] did not reactwith [HIV] virus or inhibit virus binding to CD4⁺ T cells." Thisnotation should be compared with the recent oral presentations of RonaldC. Kennedy at the 7th Annual DNA/Hybridoma Congress, San Francisco,March 1-4, 1987, as reported in Van Brunt, Bio-Technology, 5, 421-422(1987), and at the III International Conference on AcquiredImmunodeficiency Syndrome (AIDS), Washington D.C. June 1-5, 1987, (see,Abstract TH.9.5) as reported in New Scientist June 11, 1987 at page 26.In these presentations the development of polyclonal antisera againstanti-T4 antibodies was noted, as was the capacity of such antisera torecognize HIV and partially neutralize HIV infectivity in vitro. Thelatter presentation also mentioned preparation of monoclonalanti-idiotype antibody and this development is also described in Chanhet al., Proc. Nat'l. Acad. Sci. (USA), 84, 3891-3895 (June, 1987).

There continues to exist a substantial need in the art for new methodsand materials for diagnosis for the presence of HIV particles andHIV-infected cells in biological fluid and tissue specimens and also asubstantial need for new means for effecting in vivo neutralization ofthe infectivity of HIV and the development of vaccination proceduresconferring immunological protection against HIV infection.

BRIEF SUMMARY

The present invention provides purified and isolated immunologicallyactive polypeptides, preferably antibodies or chimeric antibodies orfragments thereof, and most preferably monoclonal antibodies, which arereactive with idiotypes of antibodies (preferably monoclonal antibodies)to human lymphocyte T4 protein. Such products of the invention arecapable of specific immunobinding with that portion of the HIV virionwhich is necessarily interactive with T4 surface proteins duringinfection by HIV of host cells such as human T lymphocytes and cells ofthe human nervous system. These products of the invention may becharacterized, inter alia, by their strain independent capacity toneutralize infectivity of HIV in vitro, by their specific reactivitywith HIV protein having a molecular weight of from about 60,000 to about80,000 as determined by SDS-PAGE, and by their non-reactivity with classII major histocompatability molecules associated with human immune cellsurfaces.

In one of its aspects, therefore, immunologically active products aregenerated which "respond" through specific immunobinding to HIVparticles and to the surfaces of HIV-infected cells, even though theyare generated without direct immunological reference to such surfaceproteins.

Purified and isolated polypeptides according to the invention areconspicuously suitable for use in assay procedures for the detectionand/or quantification of HIV in biological fluids wherein the detectionprocedure is based upon immunobinding between HIV particles and reactivepolypeptides (e.g., antibodies) of the invention. Moreover, antibodies,chimeric antibodies and fragments thereof developed according to theinvention are selectively immunoreactive with the surfaces ofHIV-infected cells and thus provide useful reagents for detection ofHIV-infected cells in fluid and tissue samples and for segregation ofHIV-infected cells from cell populations including both infected andnon-infected cells. As such, products of the invention will be useful indiagnostic and therapeutic procedures involving separation and/orselective destruction of HIV-infected cells.

Purified and isolated immunologically active materials of the inventionare also conspicuously suitable for use in anti-HIV treatment ofanimals, especially humans, susceptible to infection with HIV. Accordingto one such method, immunologically effective amounts of, e.g.,monoclonal antibodies of the invention, are administered to a patienteither already infected with HIV or to a patient at risk of infectionwith HIV to develop passive immunity with respect to HIV infection.According to another method, cells infected with HIV are subjected to,e.g., in vitro segregation from non-infected patient cells and thelatter may be returned to the patient.

As set forth in the following detailed description, antibody-relatedpolypeptides of the present invention are preferably obtained by meansof initial development of mono-specific antibodies (preferablymonoclonal antibodies) to the human lymphocyte T4 glycoprotein (the CD4determinant) followed by preparation of antibodies (preferablymonoclonal antibodies) to the T4 idiotypic region of antibodies formedin the initial development step.

Chimeric antibodies and fragments thereof and especially bi-specificantibodies are also products within the contemplation of the presentinvention, as are antibody-related products produced in microbial hosts,(e.g., procaryotic and eucaryotic cells in culture) which hosts aretransformed or transfected with DNA sequences encoding the desiredpolypeptide products.

As one example, with structural information in hand concerning theidiotypic regions of antibodies of the invention, it becomes possible toemploy procaryotic and eucaryotic hosts such as E. coli, yeast, insect,and mammalian cells in culture to produce useful antibody fragments(such as fab' and f(ab')₂ fragments). Moreover, it is within thecontemplation of the invention that chimeric antibodies (e.g.mouse/human antibodies) may be prepared using transformed mouse myelomacells or hybridoma cells (especially heavy chain deletion mutant cells)as production hosts. Hybrid hybridoma cell producing bi-specificantibodies having diagnostic and therapeutic uses are contemplated.Recombinant methods may also be applied to the production of HIV subunitvaccine materials. For example, monoclonal antibodies of the inventionare expected to be extremely well suited for the screening of expressionproducts of HIV DNA in transformed or transfected vial or procaryotichosts, allowing isolation of DNA encoding all or part of the amino acidsequence of naturally occurring immunologically significant HIV proteins(including glycoproteins). In suitable hosts, the presence of such DNAmay allow for high level production of vaccine materials.

In a preferred form, the invention provides antibody-relatedpolypeptides characterized as monoclonal anti-monoclonal-anti-humanlymphocyte T4 antibodies. Especially preferred are monoclonal anti-OKT4and anti-OKT4A antibodies, both of which are reactive with 60-80 Kd HIVproteins. Presently most preferred are monoclonal anti-OKT4A antibodieswhich have substantial capacity for in vitro neutralization of HIVinfectivity of multiple HIV strains and are participative incomplement-mediated cytolysis of HIV infected cells.

In another aspect, the present invention provides, for the first time,hybridoma cell lines which produce "anti-idiotypic" monoclonalantibodies specifically immunoreactive with a monoclonal antibody tohuman lymphocyte T4 protein in an antigen/antibody reaction.Illustratively, the present invention provides new murine-derivedhybridoma cell lines, JT4C8, JT4C12 and JT4C16, JT1-1F3, JT1-1F3-E5,JT1-1D7 and JT2-N15, each of which produces, as a component of thesupernatant of its growth in culture, a monoclonal antibody specificallyreactive with anti-T4 idiotype and moreover reactive with HIV virionproteins in a manner expected to allow for both in vitro and in vivoneutralization of HIV infectivity.

Hybridoma cell line JT4C8 was received for deposit with the AmericanType Culture Collection, Rockville, Md., at the facilities of the UnitedStates Department of Agriculture, Plum Island, N.Y. on Feb. 18, 1987,with A.T.C.C. Accession No. HB9385. Hybridoma cell line JT4C12 wasreceived for deposit with the American Type Culture Collection,Rockville, Md., at the facilities of the United States Department ofAgriculture, Plum Island, N.Y. on Feb. 18, 1987, with A.T.C.C. AccessionNo. HB9387. Hybridoma cell line JT4C16 was received for deposit with theAmerican Type Culture Collection, Rockville, Md., at the facilities ofthe United States Department of Agriculture, Plum Island, New York onFeb. 18, 1987, with A.T.C.C. Accession No. HB9386. Hybridoma cell lineJT1-1F3 was received for deposit at the European Collection of AnimalCell Cultures, Salisbury, Wiltshire, U.K. on Jun. 25, 1987, with ECACCAccession No. 87062501. Hybridoma cell line JT1-1F3-E5 was received fordeposit at the European Collection of Animal Cell Cultures, Salisbury,Wiltshire, U.K. on Jun. 25, 1987 with ECACC Accession No. 87062502.Hybridoma cell line JT1-1D7 was received for deposit by the FermentationResearch Institute, Ibaragi-ken, Japan on Jan. 29, 1988 with theAccession No. FERM BP-1685. Hybridoma cell line JT2-N15 was received fordeposit by the Fermentation Research Institute, Ibaragi-ken, Japan onJan. 29, 1988 with the Accession No. FERM BP-1684.

In still another of its aspects, the present invention provides forproduction of HIV subunit vaccine materials by means of well-knownaffinity purification methodologies whereby HIV protein fractions(especially those in the molecular weight range of from about 60,000 toabout 80,000 and most especially about 65,000-67,000) are isolatedthrough use of a selective immunoabsorbants prepared using antibodies ofthe invention.

Numerous aspects and advantages of the present invention will beapparent upon consideration of the illustrative examples anddescriptions of practice of the invention in the following detaileddescription thereof and the drawing, wherein: FIGS. 1, 2 and 3graphiclly represent the results of immunoreactivity testing ofantibodies of the invention with HIV and non-infected cell proteins;FIGS. 4 and 5 provide immunoblot assay results involving antibodies ofthe invention and HIV proteins; and FIGS. 6A through 6E providephotographic results of immunofluorescent staining assays on infectedand uninfected cells employing antibodies of the invention.

DETAILED DESCRIPTION

The following examples illustrate practice of the invention in theproduction of a number of hybridoma cell lines including JT4C8, JT4C12,JT4C16, JT1-1F3, JT1-1F3-E5, JT1-1D7 and JT2-N15 the isolation therefromof monoclonal antibodies to anti-CD4, and the amplification andcharacterization of such monoclonal antibodies.

More particularly, Example 1 is directed to stimulation of a murine hosttoward production of antibodies to a commercially available anti-T4monoclonal antibody, "OKT4", the fusion of spleen cells with myelomacells, the screening, cloning and growth of hybridoma cells, and theisolation of monoclonal antibodies therefrom. Example 2 relates to thecharacterization of monoclonal antibodies so produced by fluorescentimmunoassay, by Western blot assay for reactivity with HIV protein, andby screening for capacity to effect in vitro neutralizaitn of HIVinfectivity. Example 3 relates to a first procedure for development ofhybridoma cell lines capable of providing in the medium of their growthmonoclonal antibodies to the commercially available antibody "OKT4A".Example 4 relates to characterization of monoclonal antibodies soproduced by means of immunofluorescence assay, Western blot assay, invitro neutralization assay, ELISA assay, and fluorescent cell stainingassays. Example 5 relates to a second procedure for development ofhybridoma cell lines capable of providing in the medium of their growthmonoclonal antibodies to the commercially available antibody "OKT4A" andto characterization of monoclonal antibodies so produced by means ofWestern blot assay, and in vitro neutralization assay.

EXAMPLE 1

According to one aspect of the practice of the invention, hybrid tumorcell lines are produced using standard immunological techniques such asdescribed in Oi and Herzenberg, Selected Methods Cell Immunology,351-372 (1979) and Godding, "Antibody Production By Hybridomas",J.Immunol.Meth., 39, pp. 285-308 (1980). Spleen cells from mice,hyperimmunized with monoclonal anti-T4 are fused with a mouse myelomacell line in the presence of polyethylene glycol. The supernatant fromgrowth of each "hybridoma" cell culture is tested for the presence ofthe desired antibody activity. Selected hybridoma cells are cloned topropagate cell lines which produce an antibody in their growth culturesupernatant, which antibody has highly specific anti-anti-T4 activity.

A. Immunization

BALB/C mice each were subject to splenic injection with monoclonalanti-human lymphocyte T4 antibody (OKT4, Ortho Diagnostics, Rahway,N.J.).

One miligram of the lyophilized OKT4 material was brought up in 1 ml ofdistilled water. Dialysis was employed to remove and replace theoriginal phosphate buffer with 50 mM MES buffer (Sigma Chemicals), pH6.0. The material was then subjected to high pressure liquidchromatography separation on FPLC® apparatus (Pharmacia, LaboratorySeparation Division, Piscataway, N.J. 08854). Separation was carried outusing a Mono Q column under recommended procedures except that the saltgradient was changed to 0 to 0.5M NaCl. Aliquots (20 μl ) of each 0.5 mlfraction were assayed for activity using freshly collected humanlymphocytes (10⁵ cells per ml). More specifically, cells and HPLCfractions were mixed and centrifuged. The cells were then washed severaltimes and resuspended in phosphate buffered saline (PBS). 5 μg rabbitanti-mouse IgG labelled with FITC was incubated with the cells.Following centrifugation, the cell pellet was resuspended in PBS andresults were read using a fluorometric cell counter. Fractionsdisplaying highest activity were pooled, analyzed on SDS-PAGE and wererevealed to be greater than 90% pure.

Each of 4 mice was initially given a splenic injection totallingapproximately 100 μl of inoculant (approximately 1.5 μg OKT4 per mouse).Fourteen and twenty-eight days later the mice were each given boosterinjections of 100 μl inoculant.

Four days after the final booster, the mice were sacrificed and spleenswere removed aseptically and placed in petri dishes (on ice) containingDulbecco's Modified Eagle's Medium (Gibco). The spleens were trimmed offat and connective tissue, passed through 100 gauge stainless steelmesh. The resulting individual spleen cells were pelleted bycentrifugation for 10 minutes at 1000 rpm. The cell pellet was washedtwice with media (as above) and was resuspended in RPMI 1640 and thecell concentration was determined by counting in a hemocytometer in thepresence of 0.2% trypan blue.

Mouse myeloma cells NSl/1.Ag4.1 derived from Balb/c strain, were grownin RPMI 1640 medium containing 15% heat-inactivated horse serum(Pel-Freeze). The cells were pelleted by centrifugation at 1000 rpm for10 minutes and washed with RPMI 1640 containing no antibiotic. The cellconcentration was determined by counting after resuspension in the samemedium.

Spleen and NSl/1 cells were combined in a ratio of 4:1 and centrifugedat 1000 rpm for 10 minutes. The supernatant fluid was aspirated away andcell fusion was conducted at 37° C using polyethylene glycol (PEG) 1500,molecular weight 500-600. The procedure was carried out with constantgentle stirring by addition of the following, at the times indicated:1.0 ml of 50% PEG in RPMI 1640 added over one minute, with one minutestirring, 1.0 ml RPMI 1640 containing 15% horse serum over one minute;1.0 ml of RPMI 1640 containing 15% horse serum added over one minute;and 8 μl RPMI 1640 containing 15% horse serum added over 2 minutes.

The resulting fused cells were centrifuged at 1000 rpm for 10 minutes,resuspended in RPMI 1640 containing 15% horse serum and also containingpenicillin G and streptomycin at 100 units and 100 mg per ml,respectively. The cells were plated at 0.1 ml per well in 96-well platespreviously equilibrated in 5% CO₂.

Plates were incubated overnight at 37° C. in 5% CO₂.

On day two, each well received 0.1 ml of HAT medium (13.6 μg/mlhypoxanthine 0.176 μg/ml aminopterin and 3.88 μg/ml thymidine) in RPMI1640 containing 15% horse serum. This medium selectively allows spleencell NSl/1 hybrids to survive, screening out unfused NSl/1 cells orthose fused to other cells. Unfused primary spleen cells from adult micewill not survive in culture for more than a few days.

On days 3, 4, 6, 9 and 12, 0.1 ml of medium was removed from each well,and 0.1 ml of fresh HAT in RPMI 1640 containing 15% horse serum wasadded. On days 15, 19, 23 and 27, 0.1 ml of medium was removed from eachwell, and was replaced with 0.1 ml of RPMI 1640 containing 15% horseserum and only hypoxanthine and thymidine in the same concentration asabove. On day 28, culture supernatants from all wells were screened fordetection of antibody specific for OKT4.

A fluorescent-linked immunosorbent assay ("FLISA") was utilized for thedetection of hybridomas producing antibodies to OKT4. Wells of 96-wellplates were coated overnight at 37° C. with 100 μl ofcarbonatebicarbonate solution containing 3.0 μg/ml of rabbit anti-mouseIgGFc. Wells were washed ten times with PBS containing 0.05% Tween 20.The wells were blocked for 1 hour at 37° C. with 20% horse sera.Blocking agent was removed by two washings with PBS/Tween 20 as above.

Twenty microliter aliquots of culture fluid from each hybridoma well and80 μl PBS were incubated in the coated wells at room temperature for 1hour and then at 4° C. overnight. The wells were washed ten times withPBS containing 0.05% Tween 20 and blocked with 150 μl/well mouse serum(8 μg/ml), incubated for 3 hours at 37° C. and then 2 hours at 4° C.

100 μl of FITC-labelled OKT4 (0.3 μg/ml) was incubated in each wellovernight and in the dark at 4° C. Wells were washed 10 times withPBS/Tween 20 solution as above. To each well was added 200 μl of 5×10⁻⁵N NaOH and 5.6×10⁻⁴ N NH₄ OH solution. The plates were maintained atroom temperature for 15 minutes and then shaken for 1 minute. Aftertransfer to Titertek Microtitration 1×8 plates, the wells werefluorometrically (excitation 490 nm; emission 530 nm) using a CoronaElectric model MTP 100F microplate reader.

Of the 2710 wells screened, 19 were significantly positive for reactionwith OKT4. The 19 positive clones were designated JT4Cl through JT4C19.

Formal cloning of hybridoma cells obtained from the positive wells wasconducted by diluting the cells into additional wells at a ratio suchthat there was approximately 1 cell per 3 wells. Generally, formalcloning from an active well produced formal clones which appeared to besubclones of the same hybridoma cell but in several instances revealeddifferent clone populations over three generations. Subclones from thesame original well were named with the parent number and an additionalnumber (e.g., clones obtained from well JT4C7 were labeled JT4C7-1,JT4C7-2, etc.).

EXAMPLE 2

In order to characterize the antibodies produced by the positive clonesdescribed in Example 1, tests were conducted to determine relativeaffinity of monoclonal antibodies the original immunogen, the antibodieswere also screened by Western blot analysis for immunoreactivity withHIV virion proteins, and the antibodies were screened for capacity toneutralize infectivity of HIV virus.

A Relative Affinity Titrations

A fluorescent-linked assay was carried out according to the same generalprocedure employed for antibody screening in Example 1, except thatvarious dilutions of rabbit anti-mouse IgG Fc were deposited in testwells. Fluorescence results are set out in Table 1 for antibodiesderived from clones JT4Cl through JT4C-13 and JT4C16. Fluorescenceresults for various subclones of JT4C7 are set out in Table 2.

The data in Table 1 indicate that antibodies of clones JT4Cl, JT4C2 andJT4C4 are of relatively high affinity. Antibodies from clones JT4Cll andJT4C13 are of relatively lower affinity, with the antibodies of theremaining clones tested being of an intermediate affinity.

Correspondingly, Table 2 reveals that of the subclones of JT4C7, clonesJT4C7-12 and JT4C7-9 are respectively of the highest and lowest relativeaffinity in this test procedure.

                                      TABLE 1    __________________________________________________________________________    Concentration               Clone No.    Anti-Mouse Antibody               0    (μg/ml) (Blank)                    1  2  3  4  5  6  7  8  9  10 11 12 13 16    __________________________________________________________________________    10         -27  2882                       2799                          2511                             2913                                2529                                   2435                                      2601                                         2395                                            2523                                               2463                                                  1003                                                     2281                                                        1151                                                           2203    3          271  877                       803                          823                             938                                723                                   688                                      763                                         617                                            751                                               716                                                  405                                                     830                                                        497                                                           620    1          37   201                       200                          236                             128                                178                                   133                                      199                                         84 98 112                                                  92 215                                                        192                                                           272    0.3        24   31 17 106                             37 99 109                                      142                                         38 88 76 101                                                     397                                                        185                                                           263    0.1        205  14 -1 123                             23 49 53 103                                         19 49 61 16 117                                                        70 157    0.03       392  21 -82                          76 68 66 55 120                                         43 43 125                                                  76 104                                                        107                                                           202    0.01       301  15 23 105                             54 88 46 85 -51                                            40 103                                                  31 80 60 198    0.003      17   -8 -43                          86 - 14                                116                                   42 115                                         -27                                            29 126                                                  26 51 65 136    __________________________________________________________________________

                                      TABLE 2    __________________________________________________________________________    Concentration Anti-Mouse Antibody (μg/ml)    Subclone    No.  3.0            1.5 0.75                   0.37                      0.18                         0.09                            0.045                               0.022                                  0.011                                     0.005                                        0.002    __________________________________________________________________________    JT4C7-3         2364            1004                399                   337                      267                         259                            217                               206                                  246                                     215                                        161    JT4C7-12         2480            914 376                   298                      278                         262                            232                               200                                  249                                     230                                        202    JT4C7-8         2420            869 351                   291                      236                         214                            235                               201                                  217                                     188                                        208    JT4C7-9         2238            924 366                   270                      289                         214                            250                               211                                  214                                     245                                        251    JT4C7-6         2441            864 344                   250                      234                         210                            243                               210                                  203                                     239                                        235    JT4C7-4         2372            869 369                   275                      249                         243                            233                               204                                  228                                     227                                        229    JT4C7-11         2330            828 378                   290                      268                         246                            224                               211                                  245                                     228                                        227    HAT   786            475 248                   306                      241                         237                            245                               200                                  220                                     242                                        272    Medium    __________________________________________________________________________

B Western Blot Analysis

Antibodies derived from all nineteen positive clones identified inExample 1 were assayed by Western blot analysis for immunoreactivitywith proteins of the HIV virion. More specifically, HTLV-IIIB particleswere disrupted with SDS (0.1%) and dithiothriotol (0.003M) and thematerial was placed on a 7.5% polyacrylamide gel, the gel waselectrophoresed using standard procedures and materials were transferredto nitrocellulose filter paper. Filters were initially incubated with20% heat inactivated horse serum in PBS for 1 hour and then washed withPBS. Filters were then incubated with antibody supernatants of eachclone for 3 hours at room temperature with gentle shaking and thenovernight at 4° C. with gentle shaking. After washing with PBS/Tween 20,and re-blocking with 20% horse serum as above for one hour, 10 μg ofperoxidase labelled rabbit anti-mouse IgG was added and the mixture wasincubated at room temperature for 4 hours. After 10 washings withPBS/Tween 20, color was developed by standard means. Antibodies derivedfrom clones JT4C8, JT 4C12 and JT4C16 all strongly reacted with HIVprotein having a molecular weight of about 60,000 to 80,000. The abilityof these antibodies to immunoreact with HIV protein that played no partin their generation is strongly predictive of the capacity of theseantibodies to effect neutralization of HIV in vitro and in vivo.

Isotype analysis of the above-noted antibodies reactive in Western blotprocedures revealed that JT4C12 and JT4C16 antibodies were of the IgG₃isotype.

C. HIV Neutralization

Hybridoma culture supernatants were tested for HIV neutralizationcapacity in the following manner. Three day growth supernatants werediluted 1:5 in complete medium [500 ml RPMI 1640; 6 ml100×Penicillin/-Streptomycin; 6 ml 100×L-glutamine; 100 ml FCS; and, 1.2ml Polybrene Stock (1 mg/ml)]. 200 μl of the medium-diluted sample wasadded to all but two wells of a 24 well microtiter plate and two wellsreceived an equal quantity of medium alone. Additional medium was addedto one of the "medium-only" wells and each remaining well received 200μl of high titer HIV virus stock. Plates were sealed in plastic bags,incubated for 1-11/2 hours at 4° C. and allowed to return to 17° C. uponstanding for about 15 minutes. H9 cells were incubated in completemedium for 30 minutes at 37° C. at a density of 1×10⁶ cells/ml, thencentrifuged and resuspended in fresh complete medium at a density of5×10⁶ cell/ml. 200 μl of the cell suspension was added to each well(bringing the total volume to 600 μl) and the plates were incubated for1 hour at 37° C., whereupon 150 μl was transferred to a duplicate platecontaining 2.0 ml of fresh complete medium per well. Cultures wereincubated at 37° C. in a CO₂ incubator. After 4 days, the cultures weresplit and fresh complete medium was added. At day 7 of incubationsamples were prepared for neutralization screening by IFA and ReverseTranscriptase procedures. [See, Guroff et al., Nature, 316, 72-74(1985); Matthews et al., Proc. Nat'l. Acad. Sci. (USA), 83, 9709-9713(1986); and Poiesz et al., Proc. Nat'l. Acad. Sci. (USA), 77, 7415-7419(1980)]. In a first neutralization screening procedure, the results wereessentially negative or inconclusive but in a second procedure commencedconcurrently with the running of the first, antibodies from 12 of 15tested clones in the JT4Cl-19 series displayed neutralizing activity inthe IFA or RT test or both and 5 of 6 antibodies of the JT4C7 subclonestested displayed neutralization characteristics. All neutralization was"partial" in comparison to human AIDS (HTLV-IIIB) patient serum whichdisplayed 100 percent neutralization in these assays.

EXAMPLE 3

The general hybridoma forming and screening procedures of Example 1 wererepeated using the commercially available monoclonal anti-humanlymphocyte T4 antibody designated OKT4A (Ortho Diagnostics, Rahway,N.J.) which was purified using a Mono S (rather than Mono Q) column. Thespecific immunization procedure varied slightly from that of Example 1in that the initial injection was intraperitoneal and involvedapproximately 15 μg of purified antibody. The second intraperitonealinoculation was seven days later and consisted of approximately 10 μg ofpurified antibody. Thirteen days later, the final booster of about 5 μgof antibody was administered by splenic injection. Of 2090 wellsscreened, 34 were significantly positive for reaction with OKT4A, with10 of these displaying high activity (fluorescence values of about 1000or more against a "background" of approximately 200). These ten positiveclones were designated JT1-1D11, JT1-1F3, JT1-1G2, JT1-6E12, JT1- 6F12,JT2-8E9, JT3-2C4, JT3-5All, JT3-6D9, AND JT3-6E8.

EXAMPLE 4

In order to characterize the antibodies produced by the positive clonesdescribed in Example 3, tests were conducted essentially as described inExample 2. Briefly, supernatant antibodies were reactive on the Westernblot assay with HIV protein having a molecular weight of from about60,000 to about 80,000. The assays predominantly indicated reaction withan approximately 67,000 molecular weight protein, with some antibodiesshowing reactivity with a 78,000 molecular weight species. Significantlyno reactivity was noted with 41 Kd or 120 Kd fractions usuallycharacterized as the major immunologically significant HIV envelopeglycoproteins. Of the antibodies displaying the strongest reaction, two(JT1-6E12 and JT2-8E9) were of an IgM isotype and the antibody of cloneJT1-1F3 was of the IgG₁ isotype. Set out below in Table 3 are theresults of the fluorescent-linked assay for employing the JT1-1F3antibody.

                  TABLE 3    ______________________________________    Concentration Anti-    Mouse Antibody          Control    (μg/ml)    Blank     (HAT)    JT1-1F3    ______________________________________    20 μg/ml     4       754      1093    10            -37       738      1010     5            -43       756      899     2.5          -56       759      908     1.25          -2       454      634     0.625        -38       291      325     0.313         23       315      310     0.157         11       340      331    ______________________________________

Neutralization studies carried out concurrently with those of Example2(C) also indicated initial negative neutralization results and, in thesecond trial, only slight evidence of neutralization capacity on the RTassay. Nonetheless, the IgGl-secreting JT1-1F3 clone was selected forascites amplification and further antibody ELISA screening forreactivity with HIV protein.

In the first ELISA screen, varying concentrations of HTLV-IIIB protein(respectively, concentrations of 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.313,0.156, 0.078, 0.039 and 0.020 μg/ml) were coated onto microtiter plates,blocked (20% horse serum/PBS, 37° C., 2 hours), and dried overnight.Culture supernatant (20 μl with 80 μl PBS) or HAT medium control wasadded as a first antibody. After one hour of incubation at roomtemperature and storage at 4° C. overnight, peroxidase conjugated rabbitanti-mouse IgG (0.3 μg/ml in 5% horse serum/PBS) was added as the secondantibody. After 2 hours of incubation at room temperature, substrate wasadded (O-phenylenediamine, 0.5 mg/ml in McIlvan's Buffer, pH 5.5; 10 Mlof 3% hydrogen peroxide). After 20 min. of incubation at roomtemperature, t reaction was stopped with 50 μl 0.4M sulfuric andabsorbance was read at λ492-610. The test are graphically represented inFIG. 1 that HIV was detectable at levels of 1.25 μg and also thatreactivity progressively increased with up to 40 μg of virus.

The ELISA procedure was repeated on plates initially coated with varyingconcentrations of uninfected H9 cell membrane preparation [10⁶, 6×10⁵,4×10⁵, 2×10⁵, 6×10⁴, 4×10⁴, 2×10⁴, 1×10⁴, 6×20 10³, 4×10³, 2×10³cells/ml]. The results of this test procedure are graphicallyrepresented in FIG. 2 and indicate no reactivity with non-infectedpreparations.

Finally, the HIV protein ELISA was repeated using the same varyingconcentrations of protein from the HTLV-IIIB HIV variant, the HaitianHIV variant designated AL1212C (obtained from Dr. David Hall,Massachusetts General Hospital, Boston, Mass.) and the African HIVvariant designated 906 (obtained form Dr. Jerome Groopman, New EnglandDeaconess Hospital, Boston, Mass.). As graphically illustrated in FIG.3, the JT1-1F3 antibody recognized a common epitope of all three HIVvariants.

Ascites fluid derived JT1-1F3 antibody was then tested for in vitroneutalization activity as indicated by the RT assay described above andby syncitium induction. In this procedure a comparison was made betweenvarying levels of JT1-1F3 antibody (ascites fluid IgG fraction), anegative control (ascites fluid/pristane) and a positive control in theform of serum from an HTLV-IIIB-infected patient. According to thisprocedure, HIV strains IIIB, AL1212C and 906 were incubated with MabJRl-lF3, control ascites or neutralizing human serum (in a 1:5 dilutionin phosphate buffered saline) for 90 minutes at 4° C. H9 cells (5×10⁶)were then added to each well and incubated for 1 hour at 37° C. Aliquots(150 μl ) were removed from each well and added to 2.0 ml fresh medium.The cultures were split 1:1 on day 4. Reverse transcriptase activity andsyncytium induction were monitored on day 4 and day 7. The day 7 resultsof this procedure are provided in Table 4, wherein the relativesyncytium induction is indicated as follows: (-), 0/200 cells; (+/-),1-5/ 200 cells; (+), >10% cells; (++), >25% of cells; and (+++), >50% ofcells.

                                      TABLE 4    __________________________________________________________________________    JT1-1F3 NEUTRALIZATION                 HIV = HTLV-III B                                HIV = AL1212C  HIV = 906                 RT Activity/                          Syncitium                                RT Activity/                                         Syncitium                                               RT Activity/                                                        Syncitium                 % Neutralization                          Induction                                % Neutralization                                         Induction                                               % Neutralization                                                        Induction    __________________________________________________________________________      Non-infected H9                 5100/--  -     3420/--  -        4800/--                                                        -      HIV        135150/0 +++   317280/0 +++   627510/0 +++      HIV + 1 mg/ml Ab                  5730/100                          -      12030/96.2                                         -        56970/90.9                                                        -      HIV + 600 μg/ml Ab                 50700/62.4                          -     130380/58.9                                         +/-     431670/31.2                                                        -      HIV + 300 μg/ml Ab                 78330/42.0                          ++    240030/24.3                                         +       497850/20.6                                                        +/-      HIV + 50 μg/ml Ab                 132780/1.7                          +++   198690/37.4                                         +++   647670/0 ++      HIV + Ascites                 168000/0 +++   281100/11.4                                         +++   765000/0 +++      (control)      HIV + Human Serum                  5460/100                          -     197130/37.8                                         ++    688680/0 +++    __________________________________________________________________________

The results shown in Table 4 clearly demonstrate in vitro neutralizingactivity for the JT1-1F3 antibody versus all three HIV variants tested,in contrast to the neutralizing activity for infected patient serum,which was completely variant-specific, indicating recognition by themonoclonal antibody of a significant type-common epitope.

The molecular weight of the JT1-1F3 antibody reactive species wasdetermined by immunoblotting. Purified HTLV-IIIB (5 and 10 μg) wasanalyzed by SDS-PAGE and Coomasie blue/silver staining (FIG. 4, Lanes Band C). Similar aliquots were analyzed by SDS-PAGE, transferred tonitrocellulose paper and analyzed for reactivity with JT1-1F3 antibody.A single reactive species was detectable at approximately 67 kd (FIG. 4,Lanes E and F).

Reactivity of JT1-1F3 antibody with the HTLV-IIIB isolate was comparedto that with other HIV stains. Similar patterns of reactivity by ELISAwere obtained with the HTLV-IIIB, ALI2I2C and 906 strains. Furthermore,Western blot analysis with JT1-1F3 antibody and each of the three HIVstrains revealed reactivity with antigens of similar molecular weight(FIG. 5). These findings indicate that MAb JT1-1F3 reacts with relatedor identical antigens detectable in divergent HIV strains.

The above findings with JT1-1F3 antibody suggested that it might also beuseful in detecting HIV virus-infected cells. In this regard, thebinding of JT1-1F3 antibody (ascites fluid IgG fraction) to uninfectedand HIV-infected H9 cells was monitored. The extent of JT1-1F3 findingwas determined by a fluoresceinated rabbit anti-mouse IgG. As shown inFIG. 6A, there was little if any detectable binding of antibody touninfected H9 cells. In contrast, focal and diffuse binding of theantibody was detectable with HTLV-IIIB-infected H9 cells (FIG. 6B).Similar findings were obtained when using H9 cells infected with the 906and AL1212C strains (FIGS. 6C and D). This approach has been extended tohematopoietic cells from a patient with AIDS. Mononuclear cells werecollected from peripheral blood of the AIDS patient by Ficoll-Hypaqueseparation and examined for reactivity with JT1-1F3. As shown in FIG.6E, a focal and diffuse immunofluorescent staining pattern was detectedwith these cells that was similar to the findings obtained withHIV-infected H9 cells.

It is noteworthy that screening of the JT1-1F3 antibody for reactivitywith cells possessing class II major histocompatibility ("MHC") surfacecomponents (i.e., human B cell line MDl and normal peripheral bloodmononuclear cells) reveal no substantial reactivity.

Subcloning of the JT1-1F3 resulted in the selection of subclonesJT1-1F3-E5 and JT1-1D7 as respectively producing progressively higherlevels of IgG₁ antibody than JT1-1F3. Comparative neutraliziation assaydata for JT1-1F3, JT1-1F3-E5 and JT1-1D7 is set out in Table 5 below.Reactivity within the ELISA assay format for the antibodies produced bythese three hybridomas is set out in Table 6 below.

                                      TABLE 5    __________________________________________________________________________                HIV = HTLV-III B                           HIV = AL1212C                                    HIV = 906                RT Activity/                           RT Activity/                                    RT Activity/                % Neutralization                           % Neutralization                                    % Neutralization    __________________________________________________________________________    JT1-1F3    1. Non-infected H9                464/--     455/0    494/0    2. HIV      10976/0    58131/0  127317/0    3. HIV + 4 mg/ml Ab                5823/49.0  15754/73.5                                    27212/78.9    4. HIV + 2 mg/ml Ab                8561/23.0  47571/18.3                                    156653/0    5. HIV + 400 μg/ml Ab                7183/36.1  515261/11.5                                    1803291/0    JT1-1F3-E5    1. Non-infected H9                126/0       121/--  126/--    2. HIV      10990/0    33501/0  16213/0    3. HIV + 2.9 mg/ml Ab                NT*         9281/72.6                                    NT    4. HIV + 1.9 mg/ml Ab                NT         11559/65.7                                    --    5. HIV + 1 mg/ml Ab                 552/96.1  17967/46.5                                    2253/86.8    6. HIV + 500 μg/ml Ab                 775/94.0  NT       3894/76.6    JT1-1D7    1. Non-infected H9                214/0    2. HIV      10998/0    3. HIV + 500 μg/ml Ab                 177/100    4. HIV + 250 μg/ml Ab                1549/87.6    __________________________________________________________________________     *NT = Not Tested

                  TABLE 6    ______________________________________    Comparison of 3 Hybridoma on ELISA    HIV-IIB                           Control    Viral Protein             JT1-1F3  JT1-1E5  JT1-1D7                                      (Culture Media)    ______________________________________    10       1.337    1.111    1.392  0.031    5        0.914    0.887    1.118  0.023    2.5      0.996    0.831    1.081  0.022    1.25     0.499    0.278    0.523  0.011    0.63     0.221    0.122    0.176  0.020    0.31     0.070    0.075    0.060  0.015    0.16     0.048    0.049    0.036  0.004    0.08     0.027    0.027    0.031  0.012    ______________________________________

EXAMPLE 5

The hybridoma forming and screening procedures of Examples 1 and 3 wereagain repeated using OKT4A, with the following variations in theimmunization procedure. The initial immunization involvedintraperitoneal injection of 10 μg of Mono S purified OKT4A antibody;the second intraperitoneal injection (7 μg) was given seventeen dayslater; and the final, 7 μg intravenous dose was administered 18 dayslater. After fusion and screening, an IgG₁ -producing positive clone,designated JT2-N15 was selected for further study. Like JT1-1F3 and itssubclones JT1-1F3-E5 and JT1-1D7, clone JT2-N15 produced a monoclonalantibody which was reactive on the Western blot assay with HIV proteinhaving a molecular weight of about 65-67,000. Culture media from growthof JT2-N15 was positive in the ELISA assay and the results in apreliminary neutralization assay with respect to HIV-IIIB infectivityare set out in Table 7 below.

                  TABLE 7    ______________________________________    Neutralization of HIV-IIIB By JT2-N15                   RT Activity and                   % Neutralization    ______________________________________    1. Non-infected H9                     157/--    2. HIV           12194/0    3. HIV + 1.5 mg/ml Ab                     343/98.5    4. HIV + 750 μg/ml Ab                     568/96.6    ______________________________________

In further screening procedures for neutralizing activity ofanti-idiotype antibodies propagated by the ascites method, it has beenpreliminarily determined that the following protocol generates the mostactive ascites preparation. Five to eight week old mice are "primed"with 0.5 to 1.0 ml Pristane and two weeks later injected with 2 to 8×10⁶(preferably about 5×10⁶) hybridoma cells. Collection of ascites fluidscommenced two weeks after inoculation and the initial fluids collected(about 3-5 ml) displayed the highest neutralization activity. A secondcollection of ascites fluid carried out three days later produced from 8to 10 ml of fluid having lesser activity. A third collection fromsurviving animals generally provided 3-5 ml of fluid which, at times,was substantially less active than either of the materials from thefirst and second collection. Neutralization data shown for JT1-1F3 inTables 4 and 5 was based on ascites pooled from three collectionswhereas data for JT1-1F3-E5, JT 1-1D7 and JT2-N15 in Tables 5 and 7 wasbased on ascites materials pooled from first and second collectionsonly.

While the foregoing illustrative examples have been directed toprocedures involving the commercially available monoclonal antibodypreparations OKT4 and OKT4A, other commercial antibodies such as Leu3a(Becton-Dickenson, Immunocytometry Systems, Mountain View, Calif.,94039) are expected to be equally suitable for use in generatinganti-idiotype antibodies according to the invention. Equally suitableare non-commercial antibodies (preferably monoclonal antibodies)prepared by known hybridoma techniques using human T cells, human Tlymphoblast cells which express the T4 glycoprotein andrecombinant-produced human T4 protein isolates as the initial immunogen.Moreover, while antibodies produced by JT1-1F3 and its subclones andJT2-N15 are of the IgGl isotype, it is expected that antibodies ofdiffering isotypes will be equally useful. Antibodies of the IgG₂isotype, for example, may be more useful in procedures involvingcomplementmediated cytolytic reactions.

Confirmation of the operability of the procedures of the presentinvention is provided by the reports of Chanh et al., P.N.A.S. (USA),84, 3891-3895 (June, 1987) wherein it is reported that monoclonalantibody to Leu3a (designated HFl.7) was capable of in vitroneutraliziation of HIV-IIIB infectivity. However, neutralizing activityof HFl.7 has been characterized as "weak"[see Weiss, Nature, 331, p. 15(January, 1988)] and has not been demonstrated to extend to strainsother than HIV-IIIB. Moreover, unlike the anti-OKT4 and OKT4Amononoclonal antibodies of the foregoing examples, the anti-Leu3aantibody of Chanh et al. is not indicated as reactive with anyHIV-derived protein other than gp120 as shown in FIG. 4 at page 3894 ofChanh et al., supra. [See also, Dalgleish et al., The Lancet, ii,1047-1050, November 7, 1987, relating to polyclonal anti-Leu3aantibodies.]

While the foregoing examples relate to murine-derived hybridoma cellpreparations, it is within the contemplation of the invention togenerate and employ hybrid hybridomas (e.g., mouse/human) and especiallyhuman/human hybridomas prepared, for example, in a manner consistentwith Borrebaeck, TIBTECH, June, 1986, pp. 147-153; Abrams et al.,Methods in Enzymology, 121, pp. 107-119 (1986); Kozbor et al., Methodsin Enzymology, 121, pp. 120-140 (1986); Suresh et al., Methods inEnzymology, 121, pp. 210-228 (1986); and Masuho et al., Biochem. &Biophys. Res. Comm., 135(2), pp. 495-500 (1986). See, also, Klausner,"`Single Chain` Antibodies Become a Reality", Bio/Technology, 4,1042-1045 (1986), Klausner, "Stage Set For `Immunological Star Wars`",Bio/Technology, 5, 867-868 (1987) and Marx, "Antibodies Made To Order",Science, 229, 455-456 (1985).

It will be readily understood, therefore, that the above specificillustrative methodologies for the production of hybridomas and theidentification and isolation of monoclonal antibodies are not intendedto be restrictive of the scope of practice of the invention. Numerousalternative methodologies exist for achieving the same results asdemonstrated, for example, by articles appearing in Methods inEnzymology, Vol. 121, "Immunochemical Techniques, Part I", Langone etal., eds., Academic Press, Inc. (New York, 1986).

It is also within the contemplation of the invention to developimmunologically active polyeptides for use in diagnostic and therapeuticmethods of the invention by means of expression of DNA sequencesencoding therefor in suitably transformed or transfected procaryotic andeucaryotic host cells in culture.

Anti-HIV therapeutic methods of the invention will be understood tocomprise the administration of effective amounts of antibodies orantibody fragments of the invention to a patient infected with HIV or atrisk of HIV infection in order to generate passive immunity involvingneutralizing infectivity of HIV in vivo. In this regard, combination ofproducts of the invention with immunologically acceptable diluents,adjuvants and carriers is contemplated in order to form immunologicallyeffective anti-HIV therapeutic compositions.

Anti-HIV therapeutic methods of the invention within the contemplationof the invention also comprehend active immunization using biologicallyactive HIV protein fractions reactive with the monoclonal antimonoclonalanti-human lymphocytes. Such products may be obtained directly fromvirus preparations by well known affinity purification processesinvolving forming immunological reaction mixtures between HIV proteinsand antibodies of the invention, followed by isolation of the desiredprotein. As one example, the 60,000 to 80,000 molecular weight HIVprotein recognized by the JT1-1F3 in procedures of Example 4 is a primecandidate for vaccine use. The same is expected to be true forrecombinant expression products based on HIV DNA which can beimmunologically identified (and/or purified) by means of antibodies ofthe invention.

Diagnostic methods of the invention wherein polypeptide products areemployed to detect and quantify HIV particles in biological fluids suchas blood are expected to form an essential part in preliminary screeningfor patients who would benefit from passive immunization according tothe invention and in the monitoring therapeutic regimens of theinvention.

The finding that antibodies of the invention are selectively reactivewith the surfaces of HIV-infected cells is indicative of a variety ofdiagnostic and therapeutic utilities including histological screening oftissue (e.g., lymphatic cells) and fluid (e.g., blood) samples fordetection of HIV infection, possibly at early stages of infection notreadily detectable by assays based on screening for antibodies.Recognition of infected cells by antibodies of the invention allows forsegregation of such cells from cell populations comprising both infectedand non-infected cells. It is thus contemplated that blood of AIDSpatients may be subjected to extracorporeal treatment to remove orselectively kill infected lymphocytes through use of antibodies of theinvention. Moreover, within the contemplation of the invention is invivo treatment with antibodies of the invention coupled withsupplementation of circulating complement to effect cytolysis ofinfected cells. Support for the operability of such a therapeuticprotocol is provided by preliminary positive results of tests for thecapacity of JT1-1D7 antibodies to participate in the in vitro complementdependent cytolysis of HIV infected H9 cells.

Selective reactivity properties of antibodies make them good candidatesfor in vivo drug or toxin delivery to infected cells and use indevelopment of bi-specific antibodies which will include doubledeterminants allowing for, e.g., "focusing" effector T cell activity.See, e.g., Staerz et al., Proc. Nat'l. Acad. Sci. (USA), 83, 1453-1457(1986).

Based on the fact that the present invention has its foundation in theimmunological characteristics of T4 protein--which is believed toprovide the common receptor for HIV infection by all variants (e.g.,AL1212C, 906, ARC, LAV, HTLV-IIIRF, HTLV-IIIB)--rather than any specificHIV variant, it is expected that diagnostic and therapeutic methods ofthe invention will be applicable to detection and treatment of infectioninvolving all HIV variants. Use of polypeptides of the invention asdiagnostic and research tools is expected to provide additionalinformation into the nature of the interaction between HIV and hostcells. As one example, monoclonal antibodies of the invention will beuseful in identifying the precise primary, secondary and tertiarystructural conformation of region(s) of surface proteins of HIV and hostcells which are specifically and necessarily interactive in therecognition and association processes involved in HIV infection ofcells. This information, in turn, would allow for generation ofimmunologically active materials of the invention through use ofsynthetic and recombinant-produced peptide immunogens.

The foregoing detailed description has been given for clearness ofunderstanding only and no unnecessary limitations should be understoodtherefrom inasmuch as numerous modifications and variations will beexpected to occur to those skilled in the art.

What is claimed is:
 1. A method for segregating HIV-infected cells in apopulation of infected and non-infected cells, said method comprisingforming an immunological reaction mixture of said cell population and apurified and isolated immunologically active polypeptide in the form ofan antibody or chimeric antibody or antibody fragment capable ofspecific immunobinding with that portion of the HIV virion which isnecessarily interactive with T4 surface proteins during HIV infection ofhost cells and characterized by the capacity to neutralize infectivityof HIV in vitro and by specific immunoreactivity with an antibodyimmunospecific for human lymphocyte T4 and segregating infected fromnon-infected cells on the basis of the selective binding of saidpolypeptide to infected cells.
 2. A method for segregating HIV-infectedcells in a population of infected and non-infected cells, said methodcomprising forming an immunological reaction mixture of said cellpopulation and a monoclonal anti-monoclonal-anti-human lymphocyte T4antibody and segregating infected from non-infected cells on the basisof the selective binding of said antibody to infected cells.
 3. A methodfor isolating HIV proteins from a fluid sample, said method comprisingcontacting said sample with a purified and isolated immunologicallyactive polypeptide in the form of an antibody or chimeric antibody orantibody fragment capable of specific immunobinding with that portion ofthe HIV virion which is necessarily interactive with T4 surface proteinsduring HIV infection of host cells and characterized by the capacity toneutralize infectivity of HIV in vitro and by specific immunoreactivitywith an antibody immunospecific for human lymphocyte T4 to form animmunological reaction mixture including said protein and saidpolypeptide and isolating said protein from said reaction mixture.
 4. Amethod for isolating HIV proteins from a fluid sample, said methodcomprising contacting said sample with a monoclonalanti-monoclonal-anti-human lymphocyte T4 antibody to form animmunological reaction mixture including said protein and said antibodyand isolating said protein from said reaction mixture.